本文選題：結(jié)核分枝桿菌 + CFP10-ESAT6-PPE68��； 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的： 構(gòu)建結(jié)核分枝桿菌CFP10-ESAT6-PPE68融合基因原核表達(dá)質(zhì)粒，并在大腸桿菌中誘導(dǎo)表達(dá)融合蛋白。通過親和層析法分離、純化所表達(dá)的目的蛋白，將其作為一種特異性抗原，間接ELISA法檢測臨床已確診為結(jié)核�。òǚ谓Y(jié)核和肺外結(jié)核）病人的血清，統(tǒng)計(jì)分析檢測的特異性、準(zhǔn)確性和敏感性，為其在臨床上的進(jìn)一步應(yīng)用打下基礎(chǔ)。 方法： 1.以結(jié)核分枝桿菌H37Rv菌株的DNA作為模板，PCR法擴(kuò)增ESAT6和PPE68基因，利用基因拼接技術(shù)中的Gene-SOEing法擴(kuò)增ESAT6-PPE68融合基因，克隆至原核表達(dá)質(zhì)粒pET-32a(+)中，構(gòu)建重組表達(dá)質(zhì)粒pET-32a(+)-ESAT6-PPE68。再分別以H37Rv株DNA和重組質(zhì)粒pET-32a(+)-ESAT6-PPE68為模板擴(kuò)增CFP10和ESAT6-PPE68基因，Gene-SOEing法獲取目的基因CFP10-ESAT6-PPE68，克隆至pET-32a(+)載體中，構(gòu)建重組表達(dá)質(zhì)粒pET-32a(+)-CFP10-ESAT6-PPE68，并用酶切和測序鑒定。 2.將構(gòu)建成功的質(zhì)粒pET-32a(+)-CFP10-ESAT6-PPE68轉(zhuǎn)化入大腸桿菌BL21(DE3)中，IPTG誘導(dǎo)其大量表達(dá)，用Western blot鑒定重組蛋白。用親和層析法分離、純化所表達(dá)的融合蛋白。 3.將純化的融合蛋白作為一種特異性抗原，間接ELISA法檢測已臨床確診為結(jié)核�。ò�250例肺結(jié)核和66例肺外結(jié)核）病人血清，，58例正常人血清，統(tǒng)計(jì)分析檢測的特異性、準(zhǔn)確性和敏感性。 結(jié)果： 1.重組表達(dá)質(zhì)粒pET-32a(+)-CFP10-ESAT6-PPE68經(jīng)內(nèi)切酶作用后在1782bp處可見CFP10-ESAT6-PPE68目的基因片段，片段大小與預(yù)期一致�；驕y序結(jié)果顯示重組質(zhì)粒的堿基序列與理論值相符。 2.成功誘導(dǎo)表達(dá)CFP10-ESAT6-PPE68融合蛋白，SDS-PAGE分析誘導(dǎo)產(chǎn)物的相對分子量約為77KDa，包括相對分子量約20KDa的Trx條帶；相對分子量約為11KDa的CFP10；相對分子量約9KDa的ESAT6；相對分子量約為37KDa的PPE68。Western blot分析誘導(dǎo)表達(dá)蛋白可與結(jié)核分枝桿菌感染BALB/c小鼠的血清發(fā)生特異性反應(yīng)，在相對分子量約77KDa處可見明顯條帶。融合蛋白用親和層析法分離、純化后，Bandscan軟件分析目的蛋白的純度約為87.3%。 3.將純化后CFP10-ESAT6-PPE68融合蛋白作為特異性診斷抗原，以臨床確診結(jié)核病人作為金標(biāo)準(zhǔn)，58例正常人血清作為陰性對照，統(tǒng)計(jì)分析結(jié)果表明融合蛋白檢測肺結(jié)核病人和肺外結(jié)核病人的特異性分別為94.8％和98.3%；準(zhǔn)確性分別為63.6%和93.5%；敏感性分別為56.4%和89.3%。 結(jié)論： 1.將CFP10、ESAT6和PPE68三種特異性基因成功融合并插入原核表達(dá)質(zhì)粒pET-32a(+)中，并在大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)及鑒定。 2.CFP10-ESAT6-PPE68融合蛋白作為一種特異性抗原在診斷結(jié)核分枝桿菌感染方面有一定的參考價(jià)值，特別是針對肺外結(jié)核病的診斷。
[Abstract]:Objective: The prokaryotic expression plasmid of CFP10-ESAT6-PPE68 fusion gene of Mycobacterium tuberculosis was constructed and the fusion protein was induced and expressed in Escherichia coli. The expressed target protein was purified by affinity chromatography and used as a specific antigen. Indirect ELISA assay was used to detect the serum of patients with tuberculosis (including tuberculosis and extrapulmonary tuberculosis). Accuracy and sensitivity lay the foundation for further clinical application. Methods: 1. The ESAT6 and PPE68 genes were amplified by DNA from H37Rv strain of Mycobacterium tuberculosis. The fusion gene of ESAT6-PPE68 was amplified by Gene-SOEing in gene splicing technique. The ESAT6-PPE68 fusion gene was cloned into the prokaryotic expression plasmid pET-32a() and the recombinant expression plasmid pET-32a (-ESAT6-PPE68) was constructed. Then the target gene CFP10-ESAT6-PPE68 was amplified from H37Rv strain DNA and recombinant plasmid pET-32a (-ESAT6-PPE68). The target gene CFP10-ESAT6-PPE68 was cloned into pET-32a() vector. The recombinant expression plasmid pET-32a (-CFP10-ESAT6-PPE68) was constructed and identified by enzyme digestion and sequencing. 2. The constructed plasmid pET-32a (-CFP10-ESAT6-PPE68) was transformed into Escherichia coli BL21DE3, and its expression was induced by IPTG. The recombinant protein was identified by Western blot. The fusion protein was purified by affinity chromatography. 3. The purified fusion protein was used as a specific antigen. Indirect ELISA assay was used to detect the serum of 58 healthy people with clinically diagnosed tuberculosis (including 250 cases of pulmonary tuberculosis and 66 cases of extrapulmonary tuberculosis). Accuracy and sensitivity. Results: 1. The recombinant expression plasmid pET-32a (-CFP10-ESAT6-PPE68) was treated with endonuclease and the target gene fragment of CFP10-ESAT6-PPE68 was found in 1782bp, and the size of the fragment was the same as expected. The results of gene sequencing showed that the base sequence of the recombinant plasmid was consistent with the theoretical value. 2. SDS-PAGE analysis showed that the relative molecular weight of the product was about 77kDa, including the Trx band of 20KDa. CFP10 with a relative molecular weight of 11KDa, ESAT6 with a relative molecular weight of 9KDa and PPE68.Western blot with a relative molecular weight of 37KDa could induce a specific reaction with the serum of BALB/c mice infected with Mycobacterium tuberculosis. Obvious bands were observed at the relative molecular weight of about 77KDa. The fusion protein was separated by affinity chromatography and purified by Bandscan software. The purity of the target protein was about 87.3. 3. The purified CFP10-ESAT6-PPE68 fusion protein was used as the specific diagnostic antigen, and the serum of 58 normal persons was used as the negative control. The results of statistical analysis showed that the specificity of fusion protein in detecting pulmonary tuberculosis and extrapulmonary tuberculosis was 94.8% and 98.3%, the accuracy was 63.6% and 93.55.The sensitivity was 56.4% and 89.3%, respectively. Conclusion: 1. The three specific genes of CFP10 ESAT6 and PPE68 were successfully fused and inserted into the prokaryotic expression plasmid pET-32a (), and were induced and identified in E. coli BL21DE3. As a specific antigen, 2.CFP10-ESAT6-PPE68 fusion protein has a certain reference value in the diagnosis of Mycobacterium tuberculosis infection, especially for the diagnosis of extrapulmonary tuberculosis.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R378.911

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 佘茜;徐蕾;何永林;靳志棟;張丹;楊春;;結(jié)核分枝桿菌PPE68蛋白的原核表達(dá)及純化[J];中國生物制品學(xué)雜志;2010年12期

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